The General Problem to be Solved:
In sum, it is often desirable but logistically unfeasible to isolate a nucleic acid from a library because the desired nucleic acid is too severely underrepresented.
This problem arises in the screening of a nucleic acid library constructed from a plurality of heterogeneous organism forms, particularly when a desirable source organism form is disproportionately underrepresented. This problem additionally arises when a desired nucleic acid target is a relatively more rare or optionally a more unstable nucleic acid species when compared to even other nucleic acids that are derived from the same organism form.
One currently favored approach is thus to construct and screen libraries derived from a single organism source. However, the isolation of a single organism species from the rich complexity of an environmentally derived sample often requires culturing or other separation approaches to achieve homogeneity, and consequently this approach is frequently problematic and painstaking, if not unfeasible. Specifically, it has become increasingly appreciated that within the often rich complexity of an environmentally derived sample there may exist (1) a desirable source organism that possesses poorly understood culturing requirements and/or responses, (2) a desirable source organism that is problematic to culture, (4) a desirable source organism that is poorly characterized, and hence is not easily separable or distinguishable, and (5) at least two groupings of culturable but not easily separable organisms that possess incompatible culturing requirements and/or dissimilar culturing responses. Moreover, (5) the abundance of a desired nucleic acid may be prohibitively low even within a single organism species. Alternatively, (6) the abundance of a desired nucleic acid may become drastically diminished upon the subjection its source organism to culturing. Potentially still, (7) the screening process employed may require that there be an exaggerated proportional representation of a desired constituent in order for its presence to be positively identified above, e.g., the background signal. On the other hand, (8) it may be desirable to have a means to access a plurality of heterogeneous organism forms in parallel rather than in series.
The common result, nonetheless, is that—due to logistical considerations—a desired target in a library may be so overwhelmingly outnumbered by undesired components—and particularly by redundant undesired components—that it resembles a needle concealed in a forbiddingly large haystack. Accordingly, the size of the library that must be screened to expect a reasonable chance of success becomes essentially unmanageable. Thus, a particularly desired nucleic acid may be prone to virtual “loss” when subjected to conventional library construction processes and hence becomes unrecoverable during the ensuing screening processes.
In consequence, novel methods to overcome these logistical impediments are highly desirable. In particular, the screening of mixed populations of organisms is a desirable option. However, previously attempts at screening mixed populations were unfeasible if not impractical and were avoided because of the cumbersome procedures required.
A Specific Example of the Problem to be Solved:
A particular embodiment of the problem addressed by the instant invention is exemplified by, but by no means limited to, the following issue encountered in the area concerned with the search for novel microbial enzymes. Specifically, this area is concerned with the increasing demand in the research reagent, diagnostic reagent, and chemical process industries for protein-based catalysts possessing novel capabilities. At present, this need is largely addressed using enzymes purified from a variety of cultivated bacteria or fungi. However, because less than 1% of naturally occurring microbes can be grown in pure culture (Amann, 1995), alternative techniques must be developed to exploit the full breadth of microbial diversity for potentially valuable new products.
Virtually all of the commercial enzymes now in use have come from cultured organisms. Most of these organisms are bacteria or fungi. Amann et al. (Amann, 1995) have estimated the culturability of microorganisms in the environment as follows:
HabitatCulturability (%)Seawater0.001-0.1 Freshwater0.25Mesotrophic lake0.01-1.0Unpolluted esturine waters 0.1-3.0Activated sludge  1.0-15.0Sediments0.25Soil0.3
These data were determined from published information regarding the number of cultivated microorganisms derived from the various habitats indicated.
Other studies have also demonstrated that cultivated organisms comprise only a small fraction of the biomass present in the environment. For example, one group of workers recently reported the collection of water and sediment samples from the “Obsidian Pool” in Yellowstone National Park (Barns, 1994) where they found cells hybridizing to archaea-specific probes in 55% of 75 enrichment cultures. Amplification and cloning of 16S rRNA encoding sequences revealed mostly unique sequences with little or no representation of the organisms which had previously been cultured from this pool, suggesting the existence of substantial diversity of archaea with so far unknown morphological, physiological and biochemical features. Another group performed similar studies on the cyanobacterial mat of Octopus Spring in Yellowstone Park and came to the same conclusion, namely, tremendous uncultured diversity exists (Ward, 1990). Giovannoni et al. (1990) and Torsvik et al. (1990a and 1990b) have reported similar results using bacterioplankton collected in the Sargasso Sea and in soil samples, respectively. These results indicate that the exclusive use of cultured organisms in the screening for useful enzymatic or other bioactivities severely limits the sampling of the potential diversity in existence.
The screening of gene libraries from cultured samples has already proven valuable. It has recently been made clear, however, that the use of only cultured organisms for library generation limits access to the diversity of nature. The uncultivated organisms present in the environment, and/or enzymes or other bioactivities derived thereof, may be useful in industrial processes. The cultivation of each organism represented in any given environmental sample would require significant time and effort. It has been estimated that in a rich sample of soil, more than 10,000 different species can be present. It is apparent that attempting to individually cultivate each of these species would be a logistical impracticality. The alternative approach, specifically, to generate and screen a library that contains a raw and unfiltered proportional representation of all the organisms in the soil sample, likewise presents a logistical impediment. Therefore, novel methods of efficiently accessing the diversity present in the environment are highly desirable.